Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Authors

  • T.L. Buhr,

    Corresponding author
    • Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • A.A. Young,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • Z.A. Minter,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • C.M. Wells,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • D.C. McPherson,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • C.L. Hooban,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • C.A. Johnson,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • E.J. Prokop,

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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  • J.R. Crigler

    1. Dahlgren Division, CBR Concepts and Experimentation Branch (Z21) and Sensor Technology Branch (Q31), Naval Surface Warfare Center, Dahlgren, VA, USA
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Correspondence

Tony L. Buhr, Naval Surface Warfare Center, Dahlgren Division, CBR Concepts and Experimentation Branch (Z21), 4045 Higley Road, Suite 345, Dahlgren, VA 22448-5162, USA. E-mail: DLGR_NSWC_Z20@navy.mil

Abstract

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.

Introduction

Lessons learned following the 2001 anthrax attacks highlighted the need to improve biological decontamination. For example, the three chemical fumigants employed for decontamination (vaporous hydrogen peroxide, paraformaldehyde and chlorine dioxide) were effective sporicides, but damaged many materials (Holwitt et al. 2000; Anon 2003, 2004; Buhr et al. 2011, 2012). As a result, postdecontamination disposal of destroyed materials was complicated (Canter 2005). The cost of decontamination following the 2001 attacks exceeded 300 million US dollars, and the total remediation took 34 months (Buhr et al. 2012). There is a need, therefore, to develop effective decontaminants for biological agents with improved materials compatibility.

Thermal decontamination is a potential chemical-free method of remediation that may be less damaging to materials (Decker et al. 1954; Ehrlich et al. 1970; Holwitt et al. 2000). The combined effect of heat, humidity and pressure in an autoclave is an example of thermal decontamination. Higher temperatures, higher humidity and higher pressures allow for reduced sterilization times.

Previous work examining thermal spore inactivation suggests that low water content (0·6 g water g−1 dry protein), dipicolinic acid content and small acid-soluble spore proteins contribute to high heat resistance of spores (Murrell and Scott 1966; Alderton and Snell 1970; Gerhardt and Marquis 1989; Melly et al. 2002; Coleman et al. 2007; Sunde et al. 2009; Zhang et al. 2010). Spores could have additional mechanisms to resist heat such as expulsion of water from the protoplast as observed in Bacillus stearothermophilus (Prokop and Humphrey 1972). Active water removal may prevent irreversible protein aggregation and shield dry spores from harm, while the hydration achieved with humid conditions may promote irreversible protein aggregation and lead to spore death (Sunde et al. 2009). In addition, protein denaturation of heat-labile metabolic enzymes is also a generally accepted effect of thermal decontamination (Coleman et al. 2007).

The goals here were to develop test methods and justify a simulant in order to characterize and quantify the efficacy of hot, humid air inactivation of Bacillus spores dried on six different materials. Prior to testing, a spore preparation protocol useful for both B. anthracis ∆Sterne and B. thuringiensis Al Hakam was developed to eliminate spore preparation methodology as a source of experimental variability. Test methods were developed to control temperature and relative humidity while safely containing spores. The experimental design was guided by response surface methodology (RSM) in order to keep the number of tests manageable while producing meaningful data and conclusions. RSM integrates statistical design of experiments (DOE) fundamentals, regression modelling techniques and optimization methods. RSM is an iterative method widely used in industry where the aim is to use a sequence of designed experiments to obtain an optimal response (Beauregard et al. 1992; Montgomery 2009; Myers et al. 2009).

Materials and methods

Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam strains

Bacillus anthracis ∆Sterne was obtained from the Unified Culture Collection at USAMRIID (Frederick, MD, USA): Unified Culture Collection Identifier BAC1056, lot number CA062700A. Bacillus thuringiensis Al Hakam (isolated from the Iraqi Al Hakam facility) was provided by Johnathan Kiel at Brooks Air Force Base, San Antonio, TX, USA.

Spore preparation and characterization

Bacillus atrophaeus ATCC 9372 and B. cereus ATCC 4342 spores were prepared as previously described (Buhr et al. 2008). Bacillus thuringiensis Al Hakam and B. anthracis ∆Sterne sporulation medium was 0·8% nutrient broth (NB) amended with CCY salts (Stewart et al. 1981; Atrih and Foster 2001; Buhr et al. 2008) at pH 7·0. Pre-aerated and preheated sporulation medium (333 ml medium in 1-l baffled Corning flasks with filter caps) was inoculated with 105–106 spores ml−1 and incubated at 34°C with shaking (300 rev min−1) for 72 ± 2 h in a New Brunswick Scientific shaker/incubator. Cultures were amended with Tween 80 (final concentration 3%) and incubated an additional 24 ± 2 h, 34°C at 300 rev min−1 to disperse spores, specifically to address B. thuringiensis Al Hakam spore hydrophobicity. Spores were harvested and characterized via heat-resistant titres, microscopy and Coulter analysis (Buhr et al. 2008; McCartt et al. 2011; Buhr et al. 2011).

Environmental test chamber set-up and validation

Envirotronics (Grand Rapids, MI, USA) LH0010 environmental test chambers were used to control temperature and relative humidity during testing. Fifty-millilitre polypropylene conical tubes capped with 0·2-μm filter caps (TPP® TP87050; Techno Plastic Products, Trasadingen, Switzerland) were selected for hot, humid air testing in order to allow air exchange while preventing spores from escaping the conical tube and to develop a test method that reduced handling of inoculated coupons during testing. A National Institute of Standards and Technology (NIST)-certified traceable hygrometer/thermometer (11-661-18; Control Company, Friendswood, TX, USA) was positioned within a single TPP® conical tube as shown in Fig. 1. To construct the hygrometer–conical tube assembly, a hole was drilled through the conical tube bottom. The conical tube was inserted into a short piece of vinyl tubing (1·25-inch o.d. × 1-inch i.d.) and then secured using a 1·25-inch Mack washer, Parafilm® (Oshkosh, WI, USA) and electrical tape. The hygrometer probe was then inserted into the conical tube such that the measurement point was the 37·5-ml gradation marking on the conical tube and sealed with Parafilm® and electrical tape. The assembly was fitted into a piece of 1·5-inch × 2-inch polyvinylchloride and placed through the side port of the environmental chamber using a custom-cut Styrofoam plug. This assembly allowed for real-time monitoring of temperature and relative humidity inside the conical tube exactly where test coupons would be located. After assembly, temperature and relative humidity were recorded every 5 min and compared to recordings from the built-in environmental chamber sensors. Recordings continued until the hygrometer measurements matched the chamber readings, or until they remained stable for at least 15 min.

Figure 1.

Simplified diagram of the method to validate that hot, humid air would pass through 0·2-µm-filtered caps into the 50-ml conical tube containing spore-inoculated test substrates. The interior dimensions of the chamber were 49 × 38 × 54 cm. The chamber wall was 14 cm wide. The conical tubes were 3 cm diameter and 11.5 cm deep.

Coupon materials and sterilization

Square 2 × 2 cm coupons of five different test substrates and the inside surface of 50-ml TPP® polypropylene conical tubes that were used to contain the 2 × 2 cm test coupons were inoculated with >7 logs of spores. Aluminium 2024-T3 coupons painted with water-based aircraft performance coating (APC), and anti-skid material (black 60-grit anti-skid tape from No Skidding Products, Inc, Cheektowaga, NY, USA) adhered to aluminium 2024-T3 coupons (anti-skid) were purchased from the Coatings Group at the University of Dayton Research Institute (UDRI), Dayton, OH, USA. InsulFab insulation (DMS-2315-C, Type 2 Chase Facile Inc. InsulFab 315, Paterson, NJ, USA) (InsulFab) was provided by Tim Provens of Wright-Patterson Air Force Base, Dayton, OH, USA. Wiring insulation (Kapton Film Type HN, 1 mil, ref. no. 6197844-00) was purchased from Cole Parmer, Vernon Hills, IL, USA. Nylon webbing (nylon) was purchased from US Netting (Erie, PA, USA) and the ends of each coupon were cauterized to prevent fraying. Prior to testing, coupons were rinsed with 1 mega-ohm, deionized water, placed on absorbent paper in an autoclave-safe container and autoclaved on wet cycle for 30 min at 121°C. Autoclaved coupons were stored in sterile containers until used.

Response surface methodology (RSM)

As in all DOE designs, the experimental boundaries for RSM must be set by the researcher. Spore survival was the response to be modelled (i.e. the dependent variable). Time, temperature and relative humidity were the factors to be varied. Real-world limitations combined with RSM experimental design were used to select test combinations. The upper limit for temperature and relative humidity was initially set at 82°C, 90% r.h., but was adjusted to 77°C, 90% r.h. to comply with limits set by material manufacturers and because unpublished data already showed no spore survival at 82°C, 90% r.h. for 6 h. For the lower limits, the temperature was set at 60°C, 60% r.h. where spores were known to survive for multiple days. In order to fit and analyse the response surface, three equally spaced values for each factor and a centre point were tested as shown in Fig. 2. Temperatures of 140, 155 and 170°F were selected, converted to Celsius for reporting and then rounded to 60, 68 and 77°C. Each temperature–relative humidity combination was above the dew point to prevent condensation.

Figure 2.

Response surface methodology experimental design for three test factors (°C,% relative humidity, time in days). The centre point is 68°C, 75% r.h., 4 days.

Test method and design

Figure 3 shows a diagram of the decontamination test method. Prior to coupon inoculation, concentrated spores were transferred from storage (−80°C) into a water bath (50°C) for at least 30 min to thoroughly thaw spores and maintain a consistent temperature for all inoculations. These spores were then vortexed for 15–30 s and transferred to a 50-ml conical tube containing preheated (50°C) aqueous 0·1% Tween 80. The volume of 0·1% Tween 80 was set to achieve a target concentration of 2 × 108 ± 1 × 108 spores ml−1. The diluted spore inoculum was held at 50°C until coupon inoculation. At the time of inoculation, spores were vortexed again for 15–30 s and 0·1 ml of the spore inoculum was pipetted in a single drop directly onto each sterilized coupon. Inoculated coupons were left to dry overnight inside a biosafety cabinet. Dried coupons were aseptically transferred to sterile 50-ml conical tubes equipped with TPP® 0·2-μm filter caps and stored at 22 ± 3°C, ambient (40 ± 20%) relative humidity prior to testing (room temperature, RT). Polypropylene 50-ml conical tubes equipped with TPP® 0·2-μm filter caps were also directly inoculated with 0·1 ml of spores and dried (polypropylene). These conical tubes served both as a test material and as a control to determine the impact of polypropylene on spore recovery. Additionally, 0·1 ml of the spore inoculum was transferred to a solid-capped 50-ml polypropylene conical tube containing 4·9 ml of aqueous 0·1% Tween 80. These spores were kept wet and not dried (wet spores). Data from wet spores incubated at RT were compared with the original inoculum titre and represented the maximum possible number of recovered spores on a given test day. In addition, 0·1 ml of the spore inoculum was serially diluted in 0·1% Tween 80 solution and immediately plated (incubation at 37 ± 2°C for 16 ± 2 h) to quantify inoculum spore titre on the day of coupon inoculation.

Figure 3.

Step-by-step diagram of the hot, humid air decontamination method.

Table 1 shows the total number of test replicates for each test run, where a test run is defined as a single combination of temperature, time and r.h. that was tested within a single time interval. A substrate is defined as an individual coupon, polypropylene or wet sample. A replicate is defined as a substrate within a single test run which was inoculated with spores from an independent spore preparation. For each test run, five replicates of each substrate were inoculated with B. anthracis ∆Sterne spores and another five replicates of each substrate were inoculated with B. thuringiensis Al Hakam spores. A total of 10 dried coupons for each coupon type and five coupon types yielded 50 test coupons. Fifty coupons with dried spores, 10 polypropylene tubes with dried spores and 10 wet spore controls gave a total of 70 test substrates per test run. An identical set of 70 substrates were inoculated and held at RT during each test run. Thus, 70 test substrates and 70 RT substrates for a total of 140 substrates were processed during each test run. In addition, a single noninoculated coupon for each coupon type was incubated at RT to show that coupons were not contaminated (not shown in Table 1).

Table 1. Number of replicates per test run using a control driven experimental design
MaterialDried spores Wet sporesNumber for each test run
TestRTTestRT
APC10100020
Wiring insulation10100020
InsulFab10100020
Anti-skid10100020
Nylon10100020
Polypropylene1010101040
Total    140

Quantification of spore survival

After exposure to specified temperature, relative humidity and time, spores were extracted from substrates and quantified for viability using dilution plating on tryptic soy agar (TSA; Hardy Diagnostics, Santa Maria, CA, USA). Ten millilitres of preheated (37°C) extraction medium [3% tryptic soy broth (TSB; T8907, Fluka, Buchs, Switzerland), 0·25% buffered peptone water (BPW; 1.07228.0500, EMD, Darmstadt, Germany), 0·05% Tween 80 (BP338; Fisher, Pittsburgh, PA, USA), pH 7] was added to each conical tube with dried spores (polypropylene) and incubated at 37°C, 30 min for spore extraction during the first iteration of testing. During the second iteration, 10 ml of preheated (37°C) extraction medium [1% glucose (G5767; Sigma, St Louis, MO), 3% TSB, 0·25% BPW, 0·05% Tween 80, pH 7] was added to each conical tube and incubated at 37°C, 60 min for spore extraction. In both iterations, 5 ml of 2× extraction medium was added to the wet spore controls for a total of 10 ml and then incubated at 37°C. Following incubation, conical tubes were vortexed for 2 min, 22 ± 3°C on a Glasco vortexer at a setting of 70. Samples were then serially diluted and plated on TSA within 20 min of vortexing. The total time from the addition of extraction medium to plating was <60 min during the first iteration of testing and <90 min during the second iteration of testing. Plates were scored for growth after incubation at 37 ± 2°C for 16 ± 2 h. The conical tubes with the remaining 8·8 ml extraction medium, substrates and spores were also incubated at 37 ± 2°C for 16 ± 2 h to qualitatively assess the viability of all remaining spores, including those not removed from substrates. If there were no colonies on the plates and no growth in conical tubes, then the survival was scored as 0. If there was growth evident in the conical tube but no colonies observed on the plates, then this was scored as 0·1 CFU ml−1 because at least one viable spore had to be present for growth in the 8·8 ml of medium.

Spore survival calculations

Wet spore controls, incubated at 22 ± 3°C and ambient relative humidity, served as the 100% recovery reference values for calculating spore survival after hot, humid air treatment and analysis. The number of spores extracted from each spore extraction control coupon (i.e. spores dried on substrates and incubated at 22 ± 3°C and ambient relative humidity) was divided by the number from the wet spore controls to calculate spore extraction percentage. The number of surviving spores (CFU ml−1) from each hot, humid air-treated test substrate was then divided by the extraction percentage to determine the number of surviving spores in CFU ml−1. This spore concentration was then multiplied by 10 ml to give a total number of spores surviving (CFU) for each test sample. A log10 transformation of the total surviving spores was performed (log10 (total CFU + 1)).

Transmission electron microscopy (TEM)

Transmission electron microscopy analysis of untreated spores, heat-killed spores, pH-adjusted bleach-killed spores and outgrowing spores was conducted with B. anthracis ∆Sterne, B. thuringiensis Al Hakam, B. cereus ATCC 4342 and B. atrophaeus ATCC 9372 spores to provide data for simulant selection and justification. Greater than 109 spores were prepared for TEM as previously described (Anon 2007; Buhr et al. 2008). Spores were inactivated by suspending in water and incubating at 82°C for 48 h, or via treatment with pH-adjusted bleach as previously described (Anon 2007; Buhr et al. 2008). Spores were also inoculated at 5 × 107 spores ml−1 and incubated in Luria-Bertani broth at 37°C and collected at various times up to 2 h to assess germination. The heat-killed and pH 7-adjusted bleach-treated spores were checked to confirm that none survived using dilution plating on TSA.

Results

Strain selection

Preliminary tests with hot, humid air showed comparable spore kill kinetics among virulent B. anthracis Ames, attenuated B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores. Bacillus cereus ATCC 4342 spores showed higher survival rates, particularly for the wet spore controls (data not shown). This screening led to down-selection of B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

Spore Preparation and Characterization

Spore preparations were completed by four technicians. Each independent spore preparation surpassed the requirements of >108 heat-resistant spores ml−1 of sporulation medium and >95% purity by phase-contrast microscopy (Table 2). Spore size distribution data obtained from Coulter analysis showed reproducible populations of uniformly sized particles indicative of high-purity spores (Table 3). B. thuringiensis Al Hakam spores tended to agglomerate at concentrations above 109 spores ml−1 as indicated by a tail on Coulter graphs (data not shown). This was likely due to B. thuringiensis spore hydrophobicity (Doyle et al. 1984; Koshikawa et al. 1989; Husmark and Ronner 1990; Ronner et al. 1990; Faille et al. 2002).

Table 2. Titres (CFU ml−1) of heat-resistant (65°C, 30 min) spores and phase-bright percentage
 Bacillus anthracis ∆SterneBacillus thuringiensis Al Hakam
Spore titre before harvesting 4·8 × 108 ± 3·0 × 1088·2 × 108 ± 1·8 × 108
Spore titre after harvesting4·3 × 109 ± 1·1 × 1094·9 × 109 ± 1·7 × 109
Phase-bright spores (%)97·6 ± 1·698·0 ± 1·8
Table 3. Volume-equivalent spherical diameter of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores from independent preparations
 SporesSpherical diameter (μm)
B. anthracis ∆SterneCountedMeanMedianMode
Spore Prep no. 135 1571·10 ± 0·141·091·09
Spore Prep no. 246 2571·16 ± 0·261·131·12
Spore Prep no. 330 8001·13 ± 0·141·121·11
Spore Prep no. 426 9211·11 ± 0·201·101·09
Spore Prep no. 526 3451·12 ± 0·191·111·09
Spore Prep no. 635 2111·16 ± 0·181·141·13
Spore Prep no. 741 8811·14 ± 0·151·131·14
Spore Prep no. 838 0271·12 ± 0·151·111·11
Spore Prep no. 938 4081·14 ± 0·151·131·11
Spore Prep no. 1032 4981·13 ± 0·141·121·11
Spore Prep no. 1130 1271·14 ± 0·151·131·12
Spore Prep no. 1234 1981·12 ± 0·141·111·10
Combined833 0441·14 ± 0·181·121·12
B. thuringiensis Al Hakam
Spore Prep no. 176 9981·23 ± 0·171·201·19
Spore Prep no. 292641·43 ± 0·711·251·18
Spore Prep no. 312 4681·45 ± 0·711·261·19
Spore Prep no. 486 4991·22 ± 0·141·201·19
Spore Prep no. 5101 0211·21 ± 0·171·181·19
Spore Prep no. 671 0971·22 ± 0·171·191·18
Spore Prep no. 747 6901·53 ± 0·111·291·20
Spore Prep no. 827 9181·48 ± 0·641·271·19
Spore Prep no. 924 1811·63 ± 0·931·301·19
Spore Prep no. 1075 7281·20 ± 0·151·181·19
Spore Prep no. 1174 9481·21 ± 0·161·191·19
Spore Prep no. 1237 3551·43 ± 0·571·241·19
Spore Prep no. 1385 7371·20 ± 0·171·181·19
Spore Prep no. 1443 1601·26 ± 0·381·161·13
Combined1560 0001·28 ± 0·381·201·19

Figure 4 shows Coulter analysis results for two in-house spore preparations and spores prepared by an independent laboratory for B. thuringiensis using different sporulation methods. Microscopy data supplied with the independent preparation indicated 95% phase-bright spores, a purity level that was not confirmed with in-house microscopy. The broad particle size distribution shown for the independent preparation suggested large quantities of debris, and dilution plating showed a viable spore concentration of 1·1 × 108 CFU ml−1 compared with a particle count of 2·3 × 109 particles ml−1 indicated by Coulter analysis. These data supported the use of Coulter analysis as an objective method for quantifying spore debris and purity.

Figure 4.

Particle size distribution of three spore preparations, representing 4295 total particles of an in-house Bacillus anthracis ∆Sterne preparation (-------), 5,815 total particles of an in-house Bacillus thuringiensis Al Hakam preparation (– – –) and 28 834 particles of an independent laboratory's B. thuringiensis preparation (—). Number (%) is a normalized distribution representing the percentage of particles of the corresponding size.

Environmental test chamber set-up and validation

Environmental chamber temperature and relative humidity recordings were collected from the built-in environmental chamber sensors as well as NIST-certified hygrometers placed on the inside of test TPP® conical tubes. Temperature and relative humidity were measured inside conical tubes with TPP® 0·2-μm filter caps and solid-capped conical tubes (data not shown). In all experimental conditions, temperature and r.h. measured by the hygrometer inside the TPP® conical tubes with filter caps (but not those with solid caps) reached the environmental chamber set-point values within 40 min and the set conditions were maintained until the experiment was terminated. Thus, the 0·2-μm -filtered caps permitted sufficient moisture and heat exchange between the chamber and the inside of the conical tube.

Spore extraction from substrates

A single-step extraction protocol that could be used for either B. anthracis ∆Sterne or B. thuringiensis Al Hakam spores for all substrates was developed. Spore extraction with non-nutrient solutions (0·1% Tween 80 and 1% morpholino-propanesulfonic acid, pH 7) was highly variable on nylon and InsulFab (data not shown). In order to improve spore extraction efficiency, a nutrient-rich medium was used for spore extraction, similar to standardized methods ASTM E2414-05, ASTM E-2197-02, AOAC 966·04 and AOAC 2008·05 (Anon 2002, 2005; Tomasino and Hamilton 2006; Tomasino et al. 2008). The timing of cell division was monitored after extraction medium was added to B. thuringiensis Al Hakam and B. anthracis ∆Sterne spores to ensure that spores extracted from substrates were plated before cell division. The number of B. thuringiensis Al Hakam cells began to increase at 1·25 h, while B. anthracis ∆Sterne cell division was not observed until 4 h after the addition of extraction medium (Table 4). The faster germination and growth kinetics of B. thuringiensis Al Hakam determined that spores should be plated within 1 h after the addition of extraction medium, prior to cell replication.

Table 4. Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam cell division in extraction mediuma without glucose as used in the first iteration of testing and quantified with a Coulter Multisizer (cells ml−1) at the indicated time
StrainTime (h)
0·00·751·01·251·52·03·04·0
  1. a

    Spores were incubated (37°C) in extraction medium [3% TSB, 0·25% BPW, 0·05% Tween 80, pH 7].

B. anthracis ∆Sterne6·09e67·60e65·71e66·30e66·57e66·65e68·92e66·96e7
B. anthracis ∆Sterne5·02e65·08e65·48e64·92e65·09e65·35e66·24e64·67e7
B. thuringiensis Al Hakam3·22e63·21e64·31e69·40e61·89e78·64e75·50e81·04e9
B. thuringiensis Al Hakam5·01e66·72e65·06e68·46e62·29e78·45e76·42e81·37e9

Because the RSM process was iterative, further optimization of the extraction process was possible. For the second test iteration, glucose was added to the extraction medium because it is a known catabolite repressor. Addition of glucose delayed measurable cell division of B. thuringiensis Al Hakam to 2 h after the addition of extraction medium (Table 5). This allowed the timing of the spore extraction to be extended by 30 min for the second iteration of testing. The increased spore extraction time generally improved spore extraction efficiency for most substrates (Table 6).

Table 5. Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam cell division in extraction mediuma with glucose as used in the second iteration of testing and quantified with a Coulter Multisizer (cells ml−1) at the indicated time
StrainTime (h)
0·00·51·01·52·03·04·05·0
  1. a

    Spores were incubated (37°C) in extraction medium [1% glucose, 3% TSB, 0·25% BPW, 0·05% Tween 80, pH 7].

B. anthracis ∆Sterne6·31e66·90e66·71e66·54e66·80e66·11e66·33e61·55e7
B. anthracis ∆Sterne6·63e66·90e66·07e66·64e66·17e66·23e66·41e61·28e7
B. thuringiensis Al Hakam5·60e66·55e67·08e67·70e63·08e71·03e82·53e84·27e8
B. thuringiensis Al Hakam5·45e66·56e66·29e66·38e62·36e76·78e71·80e84·51e8
Table 6. Spore extraction efficiency of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores from the first iteration (test runs 1–13; no glucose present; 37°C, 30-min incubation) and second iteration (test runs 14–19; glucose present; 37°C, 60-min incubation)
B. anthracis ∆Sterne%B. thuringiensis Al Hakam%
First iteration
APC37·82APC37·75
Nylon42·15Nylon31·99
Wiring insulation47·18Wiring insulation37·37
InsulFab12·11InsulFab11·49
Anti-skid49·49Anti-skid41·65
Polypropylene75·79Polypropylene49·58
Wet100·00Wet100·00
Second iteration
APC63·67APC46·70
Nylon42·60Nylon38·59
Wiring insulation63·45Wiring insulation68·22
InsulFab30·43InsulFab33·59
Anti-skid61·35Anti-skid66·94
Polypropylene75·01Polypropylene55·24
Wet100·00Wet100·00

Hot, humid air decontamination

The log survival of spores following hot, humid air decontamination is shown in Tables 7 and 8. With the exception of the centre point (68°C, 75% r.h., 4 days), the numbers represent the arithmetic mean of five replicates. Test reproducibility at the centre point required the greatest level of confidence because partial inactivation of the spore population was anticipated. Hence, 25 replicates from five test runs were averaged per substrate for each strain at the centre point test conditions.

Table 7. Log survival of Bacillus anthracis ∆Sterne spores after exposure to hot, humid air (7·3 ± 0·3 logs of live spores inoculated (CFU) in first iteration test runs or 7·2 ± 0·0 logs inoculated (CFU) in second iteration test runs)
SubstrateTemp (°C)Relative humidity (r.h.) and time of decontamination
90% r.h.90% r.h.90% r.h.75% r.h.75% r.h.75% r.h.60% r.h.60% r.h.60% r.h.
1 day4 days7 days1 day4 days7 days1 day4 days7 days
  1. NA, Not prescribed for testing by the RSM design.

  2. a

    Denotes data from second iteration test runs.

  3. b

    Independent samples t-tests (one-sided, left tail) that showed statistically different values at α = 0·05 between inactivated test replicates and the corresponding room temperature replicates.

APC770 ± 0bNA0 ± 0bNA0 ± 0abNA6·0 ± 1·6bNA0 ± 0b
APC68NA0 ± 0abNA3·1 ± 1·3ab0·3 ± 0·2b0 ± 0abNA3·6 ± 1·8abNA
APC606·5 ± 0·6bNA0 ± 0bNA3·9 ± 0·7abNA7·2 ± 0·3NA5·3 ± 0·3b
Wiring insulation770 ± 0bNA0 ± 0bNA0 ± 0abNA5·7 ± 2·9bNA0 ± 0b
Wiring insulation68NA0 ± 0abNA0·3 ± 0·2ab0 ± 0b0 ± 0abNA0 ± 0abNA
Wiring insulation605·7 ± 3·0bNA0·2 ± 0·2bNA0 ± 0abNA7·2 ± 0·2NA4·5 ± 2·4b
Nylon770 ± 0bNA0 ± 0bNA3 ± 0·3abNA7·3 ± 0·3bNA6·3 ± 0·5b
Nylon68NA4·2 ± 0·4abNA7·1 ± 0·1ab7 ± 0·2b6·6 ± 0·2abNA7 ± 0·1aNA
Nylon607·1 ± 0·4NA6·9 ± 0·4bNA6·4 ± 0·1abNA7·3 ± 0·2NA6·9 ± 0·2
InsulFab770·6 ± 0·2bNA0 ± 0bNA0 ± 0abNA5·6 ± 2·4bNA0 ± 0b
InsulFab68NA0 ± 0abNA0·4 ± 0·2ab0·5 ± 0·6b0 ± 0abNA1·4 ± 0·8abNA
InsulFab605 ± 2·0bNA0 ± 0bNA0·4 ± 0·2abNA6·7 ± 0·5bNA5·5 ± 1·8b
Anti-skid770 ± 0bNA0 ± 0bNA0 ± 0abNA5·6 ± 1·4bNA0 ± 0b
Anti-skid68NA0 ± 0abNA3·9 ± 1·5ab0·8 ± 0·4b0·1 ± 0·2abNA2·8 ± 1·6abNA
Anti-skid604·6 ± 0·7bNA0·8 ± 0·6bNA3·3 ± 1·8abNA6·9 ± 0·3bNA5·1 ± 0·7b
Polypropylene770 ± 0bNA0 ± 0bNA0 ± 0abNA6 ± 2·6bNA0 ± 0b
Polypropylene68NA0 ± 0abNA6·2 ± 2·7ab5·9 ± 1·9b0 ± 0abNA0·3 ± 0·2abNA
Polypropylene607·1 ± 0·3NA4·5 ± 2·8bNA5 ± 1·7abNA7·2 ± 0·4NA6·4 ± 0·3b
Wet770 ± 0bNA0 ± 0bNA0 ± 0abNA0 ± 0bNA0 ± 0b
Wet68NA0 ± 0abNA0 ± 0ab0 ± 0b0 ± 0abNA0 ± 0abNA
Wet607·2 ± 0·3NA2·7 ± 1·8bNA0 ± 0abNA7 ± 0·2NA1·7 ± 1·3b
Table 8. Log survival of Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air (7·7 ± 0·2 logs of live spores inoculated (CFU) in first iteration test runs or 7·2 ± 0·0 logs inoculated (CFU) in second iteration test runs)
SubstrateTemp (°C)Relative humidity (r.h.) and time of decontamination
90% r.h.90% r.h.90% r.h.75% r.h.75% r.h.75% r.h.60% r.h.60% r.h.60% r.h.
1 day4 days7 days1 day4 days7 days1 day4 days7 days
  1. NA, Not prescribed for testing by the RSM design.

  2. a

    Denotes data from second iteration test runs.

  3. b

    Independent samples t-tests (one-sided, left tail) that showed statistically different values at α = 0·05 between inactivated test replicates and the corresponding room temperature controls.

APC770 ± 0bNA0 ± 0bNA0 ± 0abNA6·2 ± 2·2bNA0 ± 0b
APC68NA0 ± 0abNA4·2 ± 0·9ab1·2 ± 0·5b0 ± 0abNA5·4 ± 1·9abNA
APC607·3 ± 1·0NA0 ± 0bNA2·7 ± 0·2abNA7·3 ± 0·3NA7·3 ± 1·2
Wiring insulation770 ± 0bNA0 ± 0bNA0·2 ± 0·2abNA5·6 ± 2·6bNA0 ± 0b
Wiring insulation68NA0 ± 0abNA0·3 ± 0·3ab0·2 ± 0·2b0 ± 0abNA0·1 ± 0·2abNA
Wiring insulation607·3 ± 1·9NA0·2 ± 0·3bNA0 ± 0abNA7·2 ± 0·5NA7·1 ± 3·3
Nylon770·9 ± 0·5bNA0·2 ± 0·3bNA4·6 ± 0·2abNA7·3 ± 0·3bNA7·4 ± 0·5
Nylon68NA4·5 ± 0·9abNA7·1 ± 0·2a7·3 ± 0·4b6·3 ± 0·5abNA7·1 ± 0·2aNA
Nylon607·5 ± 0·4NA7·5 ± 0·4bNA7·3 ± 0·3aNA7·6 ± 0·4NA7·4 ± 0·4
InsulFab770 ± 0bNA0 ± 0bNA0·3 ± 0·2abNA5 ± 2·3bNA0 ± 0b
InsulFab68NA0 ± 0abNA2·1 ± 1·1ab3·6 ± 0·8b0 ± 0abNA4 ± 2·2abNA
InsulFab607·1 ± 3·1bNA0·5 ± 0·3bNA2 ± 1·0abNA7 ± 1·0NA7·2 ± 1·2
Anti-skid770·3 ± 0·2bNA0 ± 0bNA0 ± 0abNA6·2 ± 1·1bNA1·7 ± 0·7b
Anti-skid68NA0 ± 0abNA4·3 ± 1·0ab3·9 ± 1·4b0·2 ± 0·3abNA4·5 ± 0·6abNA
Anti-skid607·1 ± 1·6NA2·4 ± 1·2bNA4·7 ± 1·4abNA7·5 ± 0·5NA6·6 ± 1·6b
Polypropylene770 ± 0bNA0 ± 0bNA0 ± 0abNA6·5 ± 1·5bNA5·8 ± 2·9b
Polypropylene68NA0 ± 0abNA6·5 ± 1·3ab6·9 ± 1·6b0 ± 0abNA5·8 ± 2·5abNA
Polypropylene607·5 ± 0·5NA7·4 ± 2·9bNA6·9 ± 0·9aNA7·4 ± 0·5NA7·3 ± 0·6
Wet770 ± 0bNA0 ± 0bNA0 ± 0abNA0 ± 0bNA0 ± 0b
Wet68NA0 ± 0abNA0 ± 0ab0 ± 0·1b0 ± 0abNA0 ± 0abNA
Wet607·6 ± 0·4NA2·4 ± 1·3bNA0 ± 0abNA7·5 ± 0·3NA2·7 ± 1·5b

The total number of spores per substrate were determined from room temperature controls and ranged between 7·1 and 7·8 logs of spores (data not shown). Based on the results of independent samples t-tests, the number of viable spores recovered after the mildest hot, humid air test run (60°C, 60% r.h., 1 day) was statistically identical (α = 0·05) to the room temperature controls for 12 of 14 strain/substrate combinations. Spore survival for the majority of other strain/substrate/test run combinations was statistically different compared to room temperature controls at α = 0·05. Less than 1-log of viable spores was recovered for either strain on any substrate after the harshest hot, humid air test run (77°C, 90% r.h., 7 days). T-tests indicated significant differences (α = 0·20) in spore inactivation at higher temperature, higher relative humidity and longer incubation times for 36 of 42 comparisons (Table 9). A larger number of replicates would be needed to further increase the statistical confidence.

Table 9. T-test mean comparisons of hot, humid air variables and all pairs of substrates (1-sided, right tail for temperature, r.h. and time comparisons; 2-sided for substrate comparisons)
SubstrateBacillus anthracis ΔSterneBacillus thuringiensis Al Hakam
P valuesP values
Temperature: 60°C vs 77°C at 90% r.h., 1 Day
APC0·02140·0269
Wiring insulation0·18650·1681
Nylon0·01170·0442
InsulFab0·1670·1833
Anti-skid0·0660·1213
Polypropylene0·00420·1029
Wet0·01860·0342
r.h.: 90% vs 60% r.h. at 60°C, 7 days
APC0·01350·1739
Wiring insulation0·18580·1827
Nylon0·34430·6304
InsulFab0·04040·1806
Anti-skid0·0460·1568
Polypropylene0·01760·5688
Wet0·80180·3251
Time: 1 day vs 7 days at 60°C, 90% r.h.
APC0·02140·0269
Wiring insulation0·18650·1681
Nylon0·09950·5356
InsulFab0·16700·1833
Anti-skid0·06600·1213
Polypropylene0·00420·4089
Wet0·01860·0342
Materials at the centre point (68°C, 75% r.h., 4 days)
APC–Wiring insulation0·00010·0032
APC–Nylon0·00000·0007
APC–InsulFab0·03490·036
APC–Anti-skid0·05790·0347
APC–Polypropylene0·03330·0293
APC–Wet0·00010·0019
Wiring insulation–Nylon0·00000·0007
Wiring insulation–InsulFab0·03490·0342
Wiring insulation–anti-skid0·0290·0341
Wiring insulation–Polypropylene0·03330·0293
Wiring insulation–Wet1·00001·0000
Nylon–InsulFab0·00000·0007
Nylon–Anti-skid0·00000·0007
Nylon–Polypropylene0·00000·0287
Nylon–Wet0·00000·0007
InsulFab–Anti-skid0·03490·1539
InsulFab–Polypropylene0·49880·0294
InsulFab–Wet0·03490·0341
Anti-skid–Polypropylene0·03330·0295
Anti-skid–Wet0·0290·0341
Polypropylene–Wet0·03330·0293

The influence of substrate surfaces on spore survival kinetics was seen in the comparison of spore survival rates on each substrate at the RSM centre point (68°C, 75% r.h., 4 days). Table 9 shows a statistically significant difference (α = 0·05) in spore survival for 18 of 21 substrate–substrate comparisons (B. anthracis ∆Sterne) and 19 of 21 substrate–substrate comparisons (B. thuringiensis Al Hakam) at the centre point. There was <0·5 logs of spore survival for spores dried on electrostatically charged wiring insulation and wet spore controls (i.e. spores suspended in aqueous 0·1% Tween 80). However, 7 logs of spores survived on nylon and more than 5 logs of spores survived on polypropylene (oil-based materials). Survival of spores dried onto anti-skid, APC and InsulFab was between these two extremes.

Spore survival results were remarkably similar for B. anthracis ∆Sterne and B. thuringiensis Al Hakam. There were some minor differences between species at the mildest test conditions (60°C, 90% r.h., 1 day and 60°C, 60% r.h., 1 day). There were no statistical differences in B. anthracis ∆Sterne spore survival at 60°C, 90% r.h., 1 day on 3 of 7 substrates compared with the room temperature controls (Table 7), and there were no statistical differences on 5 of 7 substrates at 60°C, 60% r.h., 1 day compared with the room temperature controls (Table 7). For B. thuringiensis Al Hakam spores, there was no statistical difference in 6 of 7 substrates for the same comparisons at 60°C, 90% r.h., 1 day and 7 of 7 substrates at 60°C, 60% r.h., 1 day (Table 8). This suggested that B. thuringiensis Al Hakam spores survived slightly better on some substrates under the mildest test conditions. The most noticeable strain difference was at moderate hot, humid air conditions (68°C, 60% r.h., 4 days) on polypropylene. These conditions were effective at inactivating B. anthracis ∆Sterne spores (Table 7), but not B. thuringiensis Al Hakam spores (Table 8).

TEMs of spores

No obvious structural changes were observed in B. anthracis ∆Sterne, B. thuringiensis Al Hakam, B. cereus (ATCC 4342) and B. atrophaeus (ATCC 9372) spores that were heat-killed (Fig. 5). Likewise, Coulter analysis showed no size changes in spores after heat inactivation (data not shown).

Figure 5.

TEM micrographs of Bacillus anthracis ∆Sterne (a, b, c), Bacillus thuringiensis Al Hakam (d, e, f), Bacillus cereus ATCC 4342 (g, h, i) and Bacillus atrophaeus ATCC 9372 (J, K, L) spores. Treatments included healthy, viable spores (a, d, g, j), spores suspended in pH 7-adjusted bleach for 15 min (b, e, h, k), spores suspended in water and exposed to 82°C for 48 h (c, f) and spores germinated and outgrown in Luria-Bertani broth (i, l). Abbreviations: C = coat, OC = outer coat, IC =  inner coat, EX = exosporium, CR = crust. Black arrows point to several, but not all, breaks in the coat material (b, e, h and k). Scale lines in bottom right corner indicate 0·5 µm.

In contrast, the TEM images of spores treated with a chemical decontaminant for 15 min showed significant damage to the outer structures. The pH-adjusted bleach-treated macrobacillus spores were stripped of exosporia. The outer protein coat was damaged and often stripped as broken shards from the inner coat, which appeared intact. Exosporia and protein coats were less damaged after shorter 1-min treatments with pH-adjusted bleach (data not shown). All pH-adjusted bleach-treated B. atrophaeus spore images revealed gaps at the centre of the outer coat, suggesting that the entire coat circumference was degraded at the longitudinal centre. Both the inner coat and crust layers appeared intact around the B. atrophaeus spores, although all three outer layers were mostly separated from each other. Spore size measurements supported these observations: B. atrophaeus spore size was unchanged, while macrobacillus spores were smaller after pH-adjusted bleach treatment (Buhr et al. 2008; data not shown).

Another species comparison included TEMs of outgrown spores. B. cereus spore exosporium and coat layers were burst during germination and outgrowth, which was similar to B. anthracis (Steichen et al. 2007). Bacillus atrophaeus spore outgrowth was similar to that of B. subtilis (Santo and Doi 1974). Thus, species within the macrobacillus group manifested similar germination/outgrowth morphologies, and these differed from the microbacillus group. The single break in the exosporium and in each coat layer of outgrown B. cereus spores was markedly different than the disintegrated exosporium and broken shards of oxidizer-treated spores. Conversely, the oxidizer-generated gap in B. atrophaeus spores was observed only in the outer coat and appeared at the approximate location where the outer coat disintegrates during outgrowth.

Discussion

Test method development is a critical step towards developing and evaluating future decontaminants. This is especially true for mild technologies such as hot, humid air, where spore inactivation is explored at the limits of the decontamination technology. There was a need in the current work to carefully control and reproduce data from test runs that partially inactivated spore populations. In order to increase data confidence, three test methods were developed: a spore preparation protocol useful for both B. thuringiensis and B. anthracis; a 0·2-μm filter-cap tube method for both testing of hot, humid air and spore extraction; and a control driven experimental design. The control driven experimental design included 140 substrates (not including sterile coupon controls) per test run. This was greater than ten times the recommended maximum of 12 samples per day for the AOAC 2008.05 decontamination test method (Tomasino et al. 2008; Buhr et al. 2011). The three methods were embedded within a statistical design of experiments, specifically RSM. In addition, spores were characterized by Coulter analysis and electron microscopy.

This work established test methods and met the requirements for statistical design to show spore inactivation using hot, humid air decontamination. Such method advancements improve confidence in performance data, decrease test time, reduce test costs and support the transition of decontamination technologies from the laboratory to the field. The hot, humid air test methods were developed to translate to biosafety level 3 (BSL3) testing where safety and security requirements constrain test data output. This is highly applicable because existing and newly proposed regulations concerning possession or use of select agents or toxins limit the types of facilities and personnel that can perform BSL3 work (Anon 2011).

The test methods and experimental design permitted the examination of spore inactivation in relationship to five variables: temperature, time, r.h., substrate and strain. Temperature, time and r.h. are the three variables that can be controlled during hot, humid air decontamination. The conditions for those three variables were selected in order to explore the limits of spore inactivation for hot, humid air decontamination. The RSM-directed experimental design also required three equally spaced conditions for each of the three decontamination variables. Test conditions were selected such that spore populations would survive the mildest conditions, be partially inactivated at the mid-point conditions and be fully inactivated at the harshest conditions. The data support the selection of the experimental conditions because spores were inactivated within the RSM-directed requirements.

End-user requirements will dictate the importance of each of the three decontamination variables on hot, humid air decontamination. For example, an end-user of sensitive equipment (electronics, aircraft, etc.) will likely have a different requirement for decontamination time compared with an end-user of a ground vehicle fleet. The RSM-directed experimental design permits a broad range of end-users to apply hot, humid air decontamination technology as adjustments can be made for the three critical decontamination variables based on the requirements set by the end-user.

Materials to be decontaminated will also influence the requirements set by an end-user. Spore inactivation on different substrates was significantly different at test conditions that were between the mildest and harshest test runs. The largest number of spores survived on nylon and polypropylene, while the fewest number of spores survived in solution (wet spores) and on wiring insulation. The exosporium is the outer spore structure that imparts hydrophobic characteristics, interacts with materials and likely impacted spore survival on different materials (Doyle et al. 1984; Koshikawa et al. 1989; Husmark and Ronner 1990; Ronner et al. 1990; Charlton et al. 1999; Todd et al. 2003; Redmond et al. 2004; Faille et al. 2002; Henriques and Moran 2007; Ball et al. 2008). A hypothesis that may explain the differences in spore survival on different substrates was that porous substrates and hydrophobic, oil-based substrates repel water vapour. Thus, spore survival was highest on nylon because it was porous and hydrophobic, while spore survival on wiring insulation was lowest because this substrate had electrostatic properties. The exclusion of water vapour may have reduced heat transfer to the spores and/or reduced any effects of dissociated water molecules (H3O+ and OH). These hypothesized effects may be particularly pronounced at higher temperatures because the dissociation constant of water is more than 20-fold greater at 70°C than at 20°C (Fernandez-Prini et al. 2004).

Selection and justification of simulants is needed to achieve meaningful results and to accumulate sufficient data for confidence in a new technology, particularly for field testing (Buhr et al. 2012). Bacillus thuringiensis offers numerous advantages as a B. anthracis simulant over other candidate species (Greenberg et al. 2010). The Al Hakam strain of B. thuringiensis is genetically closer to B. anthracis than many other B. thuringiensis strains, has a fully sequenced genome and possesses no known toxin genes, and it was the suspected simulant from the Iraqi Al Hakam weapons-of-mass-destruction programme (Radnedge et al. 2003; Challacombe et al. 2007). Bacillus thuringiensis pesticidal strains (e.g. kurstaki) are commercially manufactured to favour the production of entomotoxin (Beegle and Yamamoto 1992; Avignone-Rossa and Mignone 1995). The crystal toxin, debris and fillers in such preparations vary in type and quantity to create a set of unknown variables that could impact decontamination kinetics and data interpretation. Bacillus thuringiensis Al Hakam is similar to B. anthracis because neither strain produces crystal toxin during sporulation. Bacillus thuringiensis Al Hakam spores survived similarly or slightly better than B. anthracis ∆Sterne spores under the same hot, humid air conditions on the various substrates. In total, these data support the justification of B. thuringiensis Al Hakam spores as a simulant as there is a preference that a simulant be slightly more difficult to inactivate than the agent in order to mitigate the risk of insufficient decontamination. Conversely, the use of B. thuringiensis Al Hakam may be less critical for evaluating more aggressive decontamination technologies. For example, inactivation kinetics for B. thuringiensis Al Hakam spores were similar to that of B. subtilis spores under the traumatic conditions generated by a shock-wave reaching temperatures above 226°C (McCartt et al. 2011).

Coulter and TEM analysis showed morphological similarities among macrobacillus spores before decontamination, after decontamination and during spore outgrowth. Spores inactivated with hot, humid air remained intact with no noticeable changes to spore ultrastructure. The analyses revealed no obvious clues regarding the target(s) of hot, humid air. In contrast, morphological damage was seen in the outer layers of spores, particularly macrobacillus spores, after treatment with pH-adjusted bleach. Bleach is known to be a strong oxidizer with general chemical reactivity and high heat is a denaturant. These data support the idea that bleach has pleiotropic effects and damages multiple spore surface targets; that hot, humid air is a mild decontaminant relative to pH-adjusted bleach; and that macrobacillus spores are more appropriate simulants for B. anthracis. Comparison of relevant material surfaces treated with both decontaminants would be required to confirm this assessment for end-users.

Characterization of spore morphology before decontamination, after decontamination and during spore outgrowth also supports the identification of potential spore targets for both future decontamination development and characterization of the assembly of spore structures. The macrobacillus species show very similar mechanisms of exosporium and coat shedding during outgrowth that is visibly distinct from the microbacilli species (Steichen et al. 2007; Santo and Doi 1974). The morphological damage to the exosporia and outer coat layers of pH-adjusted bleach-treated macrobacillus spores also contrasted with the specific morphological target in the outer coat of B. atrophaeus spores. Despite the differences in the outer layers of spores from macrobacillus and microbacillus species, the inner coat and cortex of all species remained intact. This suggests some common chemistry among all species that holds the coat shards together in the dormant spore. This chemistry is currently being analysed to further elucidate the mechanisms of coat assembly and disassembly.

The work here established baseline performance data for hot, humid air decontamination on clean substrates contaminated with highly purified and characterized spores. Many variables may have a significant impact on spore inactivation results. For example, differences in spore preparation conditions and spore recovery have been shown to influence the results of heat resistance testing (Alderton and Snell 1969; Cazemier et al. 2001; Melly et al. 2002). A future aim is to characterize the impact of debris on spore inactivation kinetics where the quantities and types of debris can be combined with high-purity spores under controlled conditions, thereby treating debris content as an independent, known variable (Buhr et al. 2011, 2012).

Acknowledgements

This work was supported through funding provided by the Defense Threat Reduction Agency Joint Science and Technology Office, Protection and Hazard Mitigation Capability Area (Project Number BA08PHM113). Some of the TEM work was funded by the Defense Threat Reduction Agency, Basic and Applied Sciences (Project Number AA06CBT011). We thank our colleagues at the Naval Surface Warfare Center, Dahlgren Division for technical support and Matthew J. Hornbaker and Bradford W. Gutting for editorial support. We thank Johnathan Kiel of Brooks Air Force Base for assistance and initial direction for the selection of B. thuringiensis Al Hakam as a test candidate.

Ancillary